Procedures for rapid and routine quantitative detection and counting of bacteria are important, for instance, in the practice of medicine, where bacteria counts are required for such physiological fluids as blood and urine. The prior art includes classical techniques for growing bacteria, but these are slow and require complex media. For instance, methods presently used for determining and counting bacteria in urine are culture methods which include the streak and pour plat methods and the direct methods which include microscopic counting and an additional method which depends upon the ability of organisms to oxidize nitrites to nitrates. These methods have their disadvantages. Those methods depending on a culture can be questioned because the growth of a particular bacteria will vary significantly with the particular nutrient and other environmental factors. Moreover, a significant amount of time is required for growth to occur. The direct counting methods have the disadvantage of requiring highly skilled personnel.
The prior art now includes the method disclosed in U.S. Pat. No. 3,745,090, incorporated herein by reference, entitled "Method of Detecting and Counting Bacteria in Body Fluids". The method disclosed is both rapid and routine in nature. The method depends on the fact that there is some ATP within all known living matter, including bacteria. In order to determine the bacteria count in a given urine sample, for example, a nonionic detergent is added to the urine sample to rupture all of the non-bacteria cells, placing the ATP from such cells in soluble free form. The non-bacterial ATP can then be destroyed by adding an enzyme (ATPase), such as apyrase, that will hydrolyze free ATP. The apyrase is destroyed and the bacterial cells are ruptured at the same time, freeing their ATP, by adding a strong inorganic acid such as nitric acid. The resultant suspension contains free bacterial ATP and the acid. The acid is then neutralized with a buffer or diluted with distilled water. For the purpose of the remaining test, therefore, the only effective element in the suspension is the bacterial ATP.
A luciferase-luciferin mixture is prepared in soluble form with a buffering agent to compensate for the acid in the bacterial ATP suspension if the buffer had not been previously added to the suspension, so that the effectiveness of the luciferase is not overcome. A small quantity of magnesium chloride is added. The luciferase is an enzyme which catalyzes the reaction of luciferin with ATP in the presence of Mg++, which light emission being one of the reaction end products. Luciferin is ia light emitting long chain thiol derivation.
One part of the bacterial ATP-acid solution is then mixed with one part of the luciferase-luciferin mixture. Bioluminescence will occur as a result of the reaction of the ATP with the luciferase, luciferin and the divalent metal ions. The resulting mixture is then brought into the presence of a photomultiplier system where the maximum light intensity of the bioluminescence is measured. The maximum intensity of the bioluminescence is related to the amount of ATP present which is, in turn, related to the amount of bacteria present in the original sample. A detailed description of the light measuring apparatus and techniques can be found in U.S. Pat. No. 3,756,920, issuing from a copending continuation-in-part application of U.S. Pat. No. 3,745,090 entitled "Automatic Instrument for Chemical Processing to Detect Micro-organisms in Biological Samples by Measuring Light Reactions", the disclosure of which is also incorporated by reference.
If the luciferase-luciferin mixture is present in excess the intensity of light produced is approximately proportional to the quantity of ATP present for the normal range to ATP concentrations.